Amplicon Analysis Research Articles

Metal(loid)s diffusion pathway triggers distinct microbiota responses in key regions of typical karst non-ferrous smelting assembly

Non-ferrous metal(loid)s in region with karst characteristic are highly diffusible, especially by runoff or atmospheric deposition. However, microbiota in response to the diffusing metal(loid)s is still to be understood. In this study, we focused on microbiota across metal(loid)s diffusion pathways around a non-ferrous smelting assembly. The microbial distribution and metal(loid)s-microbial interactions were analysed by 16S rRNA amplicon and multivariate statistical analysis. Although runoff and atmospheric deposition showed similar metal(loid)s diffusion contribution, different microbial compositions were revealed. The microbiota along the runoff transect (region3) was similar to those within the atmospheric deposition transect (region4), which significantly differed from those closer to the smelting assembly (region1 and region2; R2 = 0.3866, p = 0.001). Random forest model indicated the negative impacts of bioavailable metal(loid)s on microbial diversity. Proteobacteria was predominant in region1 while Actinobacteriota dominated in the other regions. Twenty abundant genera were identified in metal(loid)s rich area, such as sulfur metabolizer Sulfurifustis and metal resistant Acinetobacter. Interactions between the geochemical parameters and the dominant taxa indicated that the main drivers were Al, Sb, As and their bioavailable fractions and sulfate. This study provides understandings of microbiota patterns towards different metal(loid)s diffusion pathways around non-ferrous smelting assembly with karst characteristic.

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing.

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

Nutrient Enrichment Predominantly Affects Low Diversity Microbiomes in a Marine Trophic Symbiosis between Algal Farming Fish and Corals

While studies show that nutrient pollution shifts reef trophic interactions between fish, macroalgae, and corals, we know less about how the microbiomes associated with these organisms react to such disturbances. To investigate how microbiome dynamics are affected during nutrient pollution, we exposed replicate Porites lobata corals colonized by the fish Stegastes nigricans, which farm an algal matrix on the coral, to a pulse of nutrient enrichment over a two-month period and examined the microbiome of each partner using 16S amplicon analysis. We found 51 amplicon sequence variants (ASVs) shared among the three hosts. Coral microbiomes had the lowest diversity with over 98% of the microbiome dominated by a single genus, Endozoicomonas. Fish and algal matrix microbiomes were ~20 to 70× more diverse and had higher evenness compared to the corals. The addition of nutrients significantly increased species richness and community variability between samples of coral microbiomes but not the fish or algal matrix microbiomes, demonstrating that coral microbiomes are less resistant to nutrient pollution than their trophic partners. Furthermore, the 51 common ASVs within the 3 hosts indicate microbes that may be shared or transmitted between these closely associated organisms, including Vibrionaceae bacteria, many of which can be pathogenic to corals.

The effect of disinfectants on the microbial community on environmental healthcare surfaces using next generation sequencing

BackgroundHealthcare-associated infections are a significant economic burden and cause of avoidable morbidity and mortality within healthcare systems. The contribution of environmental contamination to healthcare-associated infection transmission has been recognized, but the mechanisms by which transmission occurs are still being investigated. The objective of this study was to characterize the microbial communities of disinfected, non-critical healthcare surfaces using next generation sequencing technology. MethodsComposite environmental surface samples were from high-touch surfaces in rooms of patients isolated for infections with multidrug-resistant organisms during their hospitalization. Information on the disinfectant product used and cleaning type (routine or terminal) was collected. 16S rRNA gene amplicon sequencing and analysis were performed. Community analysis was conducted to determine the bacterial composition and compare the detection of target pathogens by culture from 94 Contact Precaution rooms. ResultsOverall percent agreement between culture and sequence methods ranged from 52%-88%. A significant difference was observed in bacterial composition between rooms cleaned with bleach and those cleaned with a quaternary ammonium compound for composite 2 (overbed table, intravenous pole, and inner room door handle) (ANOSIM R = 0.66, P = .005) but not composite 1 (bed rails, television remote control unit, call buttons, and telephone). ConclusionsSurfaces in bleach-cleaned rooms contained a higher proportion of gram-positive microbiota, whereas rooms cleaned with quaternary ammonium compound contained a higher proportion of gram-negative microbiota, suggesting disinfectant products may impact the healthcare environment microbiome.

MP60-14 POST-BIOPSY CELL-FREE DNA IN BLOOD AS A TOOL FOR MOLECULAR TESTS IN LOCALIZED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Markers (MP60)1 Sep 2021MP60-14 POST-BIOPSY CELL-FREE DNA IN BLOOD AS A TOOL FOR MOLECULAR TESTS IN LOCALIZED PROSTATE CANCER Marinella Corbetta, Chiara Chiereghin, Ilaria De Simone, Giulia Soldà, Monica Zuradelli, Michele Giunta, Davide Maffei, Giovanni Lughezzani, Nicolò Maria Buffi, Rodolfo Hurle, Alberto Rosario Saita, Paolo Casale, Rosanna Asselta, Massimo Lazzeri, Giorgio Ferruccio Guazzoni, and Stefano Duga Marinella CorbettaMarinella Corbetta More articles by this author , Chiara ChiereghinChiara Chiereghin More articles by this author , Ilaria De SimoneIlaria De Simone More articles by this author , Giulia SoldàGiulia Soldà More articles by this author , Monica ZuradelliMonica Zuradelli More articles by this author , Michele GiuntaMichele Giunta More articles by this author , Davide MaffeiDavide Maffei More articles by this author , Giovanni LughezzaniGiovanni Lughezzani More articles by this author , Nicolò Maria BuffiNicolò Maria Buffi More articles by this author , Rodolfo HurleRodolfo Hurle More articles by this author , Alberto Rosario SaitaAlberto Rosario Saita More articles by this author , Paolo CasalePaolo Casale More articles by this author , Rosanna AsseltaRosanna Asselta More articles by this author , Massimo LazzeriMassimo Lazzeri More articles by this author , Giorgio Ferruccio GuazzoniGiorgio Ferruccio Guazzoni More articles by this author , and Stefano DugaStefano Duga More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002095.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The analysis of circulating cell-free DNA (ccfDNA) and its tumoral fraction, the circulating tumor DNA (ctDNA), represents an innovative strategy for cancer management. It can provide information on the molecular profile of patients, useful for early cancer diagnosis and monitoring. However, considering localized prostate cancer (PCa), the presence of a pseudo-capsule surrounding the organ limits the release of ccfDNA in the circulation and its tumoral fraction is under the threshold for detection. We hypothesized that during the biopsy the needle punctures performed on the organ are likely to cause a release of prostate-derived ccfDNA, suitable for further molecular tests. METHODS: We collected needle biopsies and a blood sample (3 mL) before the start of the biopsy procedure from 38 patients undergoing transperineal prostate biopsy. A second blood sample was collected at the end of the procedure, at different sampling times: for 13 patients after 60 minutes, for 19 after 120 minutes and for 6 patients 4 post-biopsy blood samples were taken (after 10, 30, 60 and 120 minutes). ccfDNA was extracted from 1 mL of plasma using the Maxwell RSC ccfDNA Plasma Kit (Promega) and quantified with a Qubit fluorometer (Thermo Fisher). The size distribution analysis was performed using a TapeStation (Agilent). Patients’ specific somatic mutations were identified with the TruSight RNA PanCancer panel (Illumina) on bioptic material and were searched in ccfDNA of the corresponding patient using a targeted sequencing (NEBNext Ultra II DNA Library Prep kit, NEB) of selected amplicons. RESULTS: We demonstrated a significant increase in the amount of ccfDNA after the biopsy procedure, both after 60 and 120 minutes (P≤0.0024). The size distribution analysis revealed the presence of longer DNA molecules, up to 1 kb, in the post-biopsy samples compared to the pre-biopsy condition. Longer molecules are probably prostate-derived and freshly released after the end of the biopsy, not yet being degraded by nucleases in the circulation. Five patient-specific somatic variants were detected with an experiment of targeted RNA-sequencing performed on needle biopsy samples. The NGS analysis of amplicons targeting the selected variants in pre- and post-biopsy ccfDNA in the corresponding patients revealed an enrichment in ctDNA (from 2.2 to 164 fold) in the post-biopsy sample CONCLUSIONS: This study opens the possibility of exploiting the enrichment of ctDNA in the post-biopsy condition for the analysis of somatic mutations/epigenetic modifications in ccfDNA for the first time in the context of localized PCa. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1047-e1047 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Marinella Corbetta More articles by this author Chiara Chiereghin More articles by this author Ilaria De Simone More articles by this author Giulia Soldà More articles by this author Monica Zuradelli More articles by this author Michele Giunta More articles by this author Davide Maffei More articles by this author Giovanni Lughezzani More articles by this author Nicolò Maria Buffi More articles by this author Rodolfo Hurle More articles by this author Alberto Rosario Saita More articles by this author Paolo Casale More articles by this author Rosanna Asselta More articles by this author Massimo Lazzeri More articles by this author Giorgio Ferruccio Guazzoni More articles by this author Stefano Duga More articles by this author Expand All Advertisement Loading ...

Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV, Decapod Penstylhamaparvovirus 1) in Commodity Red Claw Crayfish (Cherax quadricarinatus) Imported into South Korea

Freshwater crayfish, which are cultivated in aquaculture, are economically important for food and ornamental purposes. However, relatively few studies have focused on potentially pathogenic viruses in crayfish compared to in penaeid shrimp. Commodity red claw crayfish (Cherax quadricarinatus; 400 crayfish in 10 batches) and red swamp crayfish (Procambarus clarkii; 40 crayfish in 2 batches) imported into South Korea from Indonesia and China were screened by PCR to detect infectious hypodermal and hematopoietic necrosis virus (IHHNV or Decapod penstylhamaparvovirus 1). IHHNV was detected in tissue samples pooled from nine out of ten batches of red claw crayfish imported from Indonesia. Phylogenetic analysis of PCR amplicons from representative pools clustered the IHHNV strain with infectious-type II sequences commonly detected in Southeast Asian countries rather than with type III strains detected previously in whiteleg shrimp (Penaeus vannamei) cultured in South Korea. IHHNV DNA was detected most frequently in the muscle (eight batches, 66.7% samples), followed by in the hepatopancreas (five batches, 41.7% samples) and gills tissue (three batches, 25.0% samples). These data suggest that red claw crayfish could be a potential carrier of the virus and that quarantine procedures must be strengthened in South Korea to avoid importing infectious types of IHHNV in commodity crustaceans such as red claw crayfish.

Elucidating biofilm diversity on water lily leaves through 16S rRNA amplicon analysis: Comparison of four DNA extraction kits.

PremiseWithin a broader study on leaf fossilization in freshwater environments, a long‐term study on the development and microbiome composition of biofilms on the foliage of aquatic plants has been initiated to understand how microbes and biofilms contribute to leaf decay and preservation. Here, water lily leaves are employed as a study model to investigate the relationship between bacterial microbiomes, biodegradation, and fossilization. We compare four DNA extraction kits to reduce biases in interpretation and to identify the most suitable kit for the extraction of DNA from bacteria associated with biofilms on decaying water lily leaves for 16S rRNA amplicon analysis.MethodsWe extracted surface‐associated DNA from Nymphaea leaves in early stages of decay at two water depth levels using four commercially available kits to identify the most suitable protocol for bacterial extraction, applying a mock microbial community standard to enable a reliable comparison of the kits.ResultsKit 4, the FastDNA Spin Kit for Soil, resulted in high DNA concentrations with better quality and yielded the most accurate depiction of the mock community. Comparison of the leaves at two water depths showed no significant differences in community composition.DiscussionThe success of Kit 4 may be attributed to its use of bead beating with a homogenizer, which was more efficient in the lysis of Gram‐positive bacteria than the manual vortexing protocols used by the other kits. Our results show that microbial composition on leaves during early decay remains comparable and may change only in later stages of decomposition.

Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.

Intramolecular recombination enables the formation of hepatitis B virus (HBV) cccDNA in mice after HBV genome transfer using recombinant AAV vectors

The mouse is not a natural host of hepatitis B virus (HBV) infection and - despite engraftment of hepatocytes with the HBV receptor - does not support formation of HBV covalently closed circular (ccc) DNA serving as a template for viral transcription and permitting persistent infection. In a recent study, cccDNA formation in mouse hepatocytes has been described following an HBV genome delivery by a recombinant, adeno-associated virus vector (rAAV) (Lucifora et al., 2017). The integrity of HBV cccDNA, its origin and functionality, however, remained open. In this study, we investigated the identity, origin, and functionality of cccDNA established in mice infected with rAAV carrying 1.3-fold overlength HBV genomes. We show that replication of HBV genotypes A, B, C and D can be initiated in mouse livers, and that cccDNA derived from all genotypes is detected. Restriction enzyme and exonuclease digestion as well as sequencing analysis of cccDNA amplicons revealed authentic HBV cccDNA without any detectable alteration compared to cccDNA established after HBV infection of human liver cells. Mouse livers transduced with a core protein-deficient HBV using rAAV still supported cccDNA formation demonstrating that the genesis of cccDNA was independent of HBV replication. When mice were infected with an rAAV-HBV1.3 carrying premature stop codons in the 5′ but not in the 3′ core protein open reading frame, the stop codon was partially replaced by the wild-type sequence. This strongly indicated that intramolecular recombination, based on >900 identical base pairs residing at the both ends of the HBV1.3 transgene was the origin of cccDNA formation. Accordingly, we observed a constant loss of cccDNA molecules from mouse livers over time, while HBeAg levels increased over the first two weeks after rAAV-HBV1.3 infection and remained constant thereafter, suggesting a minor contribution of the cccDNA molecules formed to viral transcription and protein expression. In summary, our results provide strong evidence that intramolecular recombination of an overlength, linear HBV genome, but not HBV genome recycling, enables cccDNA formation in rAAV-HBV mouse models.

Microbial Communities of Hydrothermal Guaymas Basin Surficial Sediment Profiled at 2 Millimeter-Scale Resolution.

The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin.

Maternal supplementation with phytogenic additives influenced the faecal microbiota and reproductive potential in sows

Sows undergo physiological stress during gestation and lactation, potentially leading to enteric dysbiosis and reduced reproductive potential. Phytogenic additives (PFs) may improve performance via their antioxidant, anti-inflammatory and antimicrobial properties. This study determined whether the provision of a gestation/lactation diet containing PAs would alter the gastrointestinal microbiota of sows and their piglets, and improve performance. Sows received a commercial diet throughout gestation and lactation (CTR; n = 64), a commercial diet throughout gestation and a diet containing PAs in lactation (CTR-PA; n = 63) or a commercial diet containing PAs in gestation and lactation (PA; n = 90). Sows were weighed and backfat recorded after mating and at entry and exit from the farrowing house and piglets were weighed on days 1 and 21 of life. Faecal samples collected from sows at farrowing house entry and piglets at 21 and 35 d were subjected to 16 S rRNA gene amplicon analysis. The addition of PAs to sow diets resulted in more piglets born (P = 0.03), however, it did not improve the number of liveborn piglets (P = 0.14). There were no differences in sow weight, P2 backfat depth or lactation feed intake observed. PAs had no effect on piglet weight or survival to weaning but did alter the faecal microbiota of sows, and this change was observed in piglets at 21 and 35 d. PA supplementation to sows has the potential to increase litter size, while also potentially influencing gastrointestinal tract health of the sow and piglets reared.

Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

Farklı Bakteriyel Hastalıklara Maruz Kalmış Gökkuşağı Alabalıklarında Karaciğer ve Beyin Dokularında AChE ve BChE Enzim Aktivite Seviyesi Farklılıkları

In this study, symptomatic fish samples were taken from rainbow trout farms. Isolation and identification of agents isolated from fish samples were made. DNA isolations from different purified colonies were carried out with the mericon bacterial DNA kit. Real-Time PCR procedure was performed by using universal bacterial primers. Molecular identifications were performed by blasting the nucleotides obtained by sequence analysis of PCR amplicons. Spectrophotometric measurements were performed at 412 nm wavelengths for AChE activity and 412 nm for BChE activity from liver and brain tissues of fish samples. The activity differences of different disease factors among themselves and according to the control group were examined. As a result of the study, isolation and identification of Bacillus subtilis, Lactococcus garvieae and Staphylococcus epidermidis from 5 different farms were performed. Over 98% similarity was observed as a result of sequencing analysis of the isolates. In this study, it was observed that three different bacteria isolated from trout farms suppressed AChE and BChE enzyme activities in both tissues of trout.

Recurrent STAT3 Mutations in the Lymphocytes of Pure Red Cell Aplasia without T-Cell Large Granular Lymphocytic Leukemia

▪Background: Activating somatic mutations of STAT3 are one of the most frequently recognized genetic alterations in T-cell lymphoma including T-cell large granular lymphocytic leukemia (T-LGLL). T-LGLL is associated with certain proportions of patients (pts) with acquired aplastic anemia (AA) and/or myelodysplastic syndromes (MDS). Pure red cell aplasia (PRCA), especially acquired PRCA, is a syndrome defined by an anemia with marked reduction or absence of erythroid production associated with immunological mechanisms. PRCA is classified into primary and secondary types including T-LGLL. Previous reports have described that T-LGLL with STAT3 mutations was closely associated with PRCA. However, it is yet to be clear whether STAT3-mutated T cells would be involved in the pathogenesis of other cytopenias, such as AA, MDS, primary PRCA or secondary PRCA without (w/o) T-LGLL. In this study, we analyzed the profiles of STAT3 gene mutations in bone marrow failure syndrome (BMFS)s including AA/MDS and PRCA.Methods: Peripheral blood samples were collected from 94 patients with BMFSs, including AA, MDS, AA/PNH, and PRCA with or w/o T-LGLL. When possible, T-cell receptor (TCR) Vβ repertoire of the patients was determined by flow cytometry. DNA was extracted from mononuclear cells (MNC), and STAT3 Y640F and D661Y, two hot spot mutations, were detected with sensitive allele specific-PCR (AsPCR) methods. Then we performed deep amplicon sequencing analysis of STAT3 in PRCA pts. CD8+ T cells were sorted if possible, and also subjected to the analyses.Results: The recruited pts included 38 AA, 20 MDS, 7 AA/PNH, 11 PRCA with T-LGLL, and 18 PRCA w/o T-LGLL. Among PRCA pts w/o apparent T-LGLL, 10 had primary PRCA and 8 had secondary PRCA including 2 with thymoma. The clinical characteristics of PRCA are shown in Table 1. TCR Vβ repertoire analysis were performed in 8 PRCA w/o T-LGLL and 5 PRCA with T-LGLL. All pts with PRCA associated with T-LGLL showed restricted Vβ repertoire. On the other hand, only 1pt showed monoclonal Vβ pattern in PRCA w/o TLGLL. AsPCR did not detect Y604F or D661Y mutations in any of 65 pts with AA, MDS, or AA/PNH. In contrast, 2 pts with PRCA and 4 pts with PRCA-T-LGLL were positive for either of the mutations, suggesting STAT3 mutations may be unique to PRCA pts. Further analysis with deep amplicon analysis in the 29 PRCA pts revealed STAT3 mutations in 11 pts (61%) of PRCA w/o T-LGLL, which included 6 pts with primary PRCA and 5 pts with secondary PRCA associated with autoimmune diseases (n=2), 2 thymoma (n=2) and thymic cancer (n=1). The median variant allele frequency of STAT3 mutations was 2.3% (0.2-48.9). N538Y and I653V were most frequent mutations in PRCA w/o T-LGLL. Eight pts (73%) with T-LGLL-associated PRCA were also positive for STAT3 mutation. Thirty-nine % and 70% of STAT3 mutation were found in SH2 domain in PRCA w/o T-LGLL and PRCA with T-LGL, respectively. There was no difference in clinical features between pts with and w/o STAT3 mutations.Discussion: The high frequency of STAT3 mutations in PRCA pts regardless of etiology and the absence of the mutation in pts with AA/MDS suggest that STAT3 mutations may serve as a molecular marker of PRCA. The discordant results with regard to AA/MDS pts from a previous report which detected STAT3 mutations in 4.9% of AA/MDS patients by AsPCR methods (Jerez et al. BLOOD 2013) may be due to the difference in ethnic backgrounds of the pts studied. Given very low frequencies of STAT3 mutations in AA and MDS that were revealed by several previous studies, JAK/STAT signaling systems in the axis of STAT3 mutations may have unique roles in the pathogenesis of PRCA.Conclusion: STAT3 mutations are frequently detected in pts with PRCA even in pts w/o T-LGLL and the JAK/STAT pathway may therefore be therapeutic targets of PRCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rapid Increase of SARS-CoV-2 Variant B.1.1.7 Detected in Sewage Samples from England between October 2020 and January 2021.

ABSTRACTSARS-CoV-2 variants with multiple amino acid mutations in the spike protein are emerging in different parts of the world, raising concerns regarding their possible impact on human immune response and vaccine efficacy against the virus. Recently, a variant named lineage B.1.1.7 was detected and shown to be rapidly spreading across the UK since November 2020. As surveillance for these SARS-CoV-2 variants of concern (VOCs) becomes critical, we have investigated the use of environmental surveillance (ES) for the rapid detection and quantification of B.1.1.7 viruses in sewage as a way of monitoring its expansion that is independent on the investigation of identified clinical cases. Next-generation sequencing analysis of amplicons synthesized from sewage concentrates revealed the presence of B.1.1.7 mutations in viral sequences, first identified in a sample collected in London on 10 November 2020 and shown to rapidly increase in frequency to >95% in January 2021, in agreement with clinical data over the same period. We show that ES can provide an early warning of VOCs becoming prevalent in the population and that, as well as B.1.1.7, our method can detect VOCs B.1.351 and P.1, first identified in South Africa and Brazil, respectively, and other viruses carrying critical spike mutation E484K, known to have an effect on virus antigenicity. Although we did not detect such mutation in viral RNAs from sewage, we did detect mutations at amino acids 478, 490, and 494, located close to amino acid 484 in the spike protein structure and known to also have an effect on antigenicity.IMPORTANCE The recent appearance and growth of new SARS-CoV-2 variants represent a major challenge for the control of the COVID-19 pandemic. These variants of concern contain mutations affecting antigenicity, which raises concerns on their possible impact on human immune response to the virus and vaccine efficacy against them. Here, we show how environmental surveillance for SARS-CoV-2 can be used to help us understand virus transmission patterns and provide an early warning of variants becoming prevalent in the population. We describe the detection and quantification of variant B.1.1.7, first identified in southeast England in sewage samples from London (UK) before widespread transmission of this variant was obvious from clinical cases. Variant B.1.1.7 was first detected in a sample from early November 2020, with the frequency of B.1.1.7 mutations detected in sewage rapidly increasing to >95% in January 2021, in agreement with increasing SARS-CoV-2 infections associated with B.1.1.7 viruses.

Metagenomic analysis of gut microbiota reveals its role in trimethylamine metabolism in heart failure

BackgroundWe had previously reported an increase in trimethylamine N-oxide (TMAO) levels in patients with both compensated and decompensated heart failure (HF) and alteration in gut microbiota composition using 16S rRNA gene amplicon analysis. Although a metagenome-wide analysis showed that choline-TMA lyase levels increased in HF patients, which TMA generation pathway from choline, carnitine, or betaine contributes to the increase in TMAO levels in HF needs to be elucidated. MethodsWe conducted a metagenome-wide shotgun sequencing analysis of gut microbiota and measured the TMAO levels in plasma of 22 HF patients during the compensated phase and 11 age-, sex-, and comorbidity-matched control subjects, whose gut microbiota compositions were reported in a previous 16S rRNA-based analysis. ResultsThe abundance of cntA/B was positively correlated with TMAO, especially in HF patients, whereas that of cutC/D or betaine reductase was not correlated either in controls or HF patients. The abundance of cntA/B was mainly derived from the genera Escherichia and Klebsiella either in controls or HF patients. ConclusionTMAO levels in plasma depend on the abundance of cntA/B in HF. Although it is difficult to exclude the involvement of confounding factors, microbial dysbiosis connecting the abundance of cntA/B in the gut and the increase of TMAO in plasma can be a therapeutic target for HF.

Exploring untapped potential of Streptomyces spp. in Gurbantunggut Desert by use of highly selective culture strategy

Streptomycetes have been, for over 70 years, one of the most abundant sources for the discovery of new antibiotics and clinic drugs. However, in recent decades, it has been more and more difficult to obtain new phylotypes of the genus Streptomyces by using conventional samples and culture strategies. In this study, we combined culture-dependent and culture-independent approaches to better explore the Streptomyces communities in desert sandy soils. Moreover, two different culture strategies termed Conventional Culture Procedure (CCP) and Streptomycetes Culture Procedure (SCP) were employed to evaluate the isolation efficiency of Streptomyces spp. with different intensities of selectivity. The 16S rRNA gene amplicon analysis revealed a very low abundance (0.04–0.37%, average 0.22%) of Streptomyces in all the desert samples, conversely the percentage of Streptomyces spp. obtained by the culture-dependent method was very high (5.20–39.57%, average 27.76%), especially in the rhizospheric sand soils (38.40–39.57%, average 38.99%). Meanwhile, a total of 1589 pure cultures were isolated successfully, dominated by Streptomyces (29.52%), Microvirga (8.06%) and Bacillus (7.68%). In addition, 400 potential new species were obtained, 48 of which belonged to the genus Streptomyces. More importantly, our study demonstrated the SCP strategy which had highly selectivity could greatly expand the number and phylotypes of Streptomyces spp. by almost 4-fold than CCP strategy. These results provide insights on the diversity investigation of desert Streptomyces, and it could be reference for researchers to bring more novel actinobacteria strains from the environment into culture.

Microscopic Colitis Patients Possess a Perturbed and Inflammatory Gut Microbiota.

Microscopic colitis (MC), an inflammatory disease of the colon, is characterized by chronic non-bloody diarrhea with characteristic inflammation and for some, collagen deposits in mucosal biopsies. The etiology of MC is unclear, although previous findings implicate luminal factors and thus the gut microbiome. However, the relationships between fecal microbiota and MC are relatively unexplored. Stool microbiota of MC (n = 15) and healthy controls (HC; n = 21) were assessed by 16S rRNA V4 amplicon sequencing and analysis performed in QIIME. Gut microbiota functions were predicted using Piphillin and inflammatory potential assessed using an in vitro HT29 colonocyte cell assay. MC patient fecal microbiota were less diverse (Faiths index; p < 0.01) and compositionally distinct (PERMANOVA, weighted UniFrac, R2 = 0.08, p = 0.02) compared with HC subjects. MC microbiota were significantly depleted of members of the Clostridiales, enriched for Prevotella and more likely to be dominated by this genus (Chi2 = 0.03). Predicted pathways enriched in MC microbiota included those related to biosynthesis of antimicrobials, and sphingolipids, to glycan degradation, host defense evasion, and Th17 cell differentiation and activation. In vitro, exposure of cultured colonocytes to cell-free products of MC patient feces indicates reduced gene expression of IL-1B and occludin and increased GPR119 and the lymphocyte chemoattractant CCL20. MC gut microbiota are distinct from HC and characterized by lower bacterial diversity and Prevotella enrichment and distinct predicted functional pathways. Limited in vitro experiments indicate that compared with cell-free products from healthy fecal microbiota, MC microbiota induce distinct responses when co-cultured with epithelial cells, implicating microbiota perturbation in MC-associated mucosal dysfunction.

Exposure to maternal feces in lactation influences piglet enteric microbiota, growth, and survival preweaning.

It is known that gilt progeny performance is reduced compared with sow progeny. Previous research suggests that the presence of maternal feces in early life improves the health and survival of offspring. Therefore, we aimed to determine whether contact with feces from multiparous (MP) sows would improve the growth and survival of piglets born and reared on primiparous (P1) sows and if so, whether these differences are associated with the gut microbiota. Four treatments were applied for 10 days: Donor (n = 29) piglets had limited access to maternal feces as, each morning, sow feces were removed and placed in the crate of a P1 sow (P1-FT; n = 30 piglets) and P1-Con (n = 29) and MP-Con (n = 33) piglets had access to their own mothers’ feces. All piglets were weighed on days 1, 3, 10, and 18. Fecal samples were collected from a subset of sows (n = 10/treatment) 3 days post farrow and from two female piglets/litter on days 10 and 18 (n = 20/treatment) and subject to 16S rRNA amplicon analysis. Escherichia, Clostridium, Campylobacter, and Treponema were more abundant in MP sows, while P1 sows had a higher abundance of Lactobacillus and Prevotella. At 10 days, P1 progeny fecal microbiota differed, and growth and survival were reduced when compared with MP progeny. No treatment effect was observed for P1-FT piglets (P > 0.05). Donor piglets had a different fecal microbiota and improved weight and survival then all other treatments (P < 0.05). Overall, the removal of sow feces from the farrowing crate improved piglet microbiota development, growth, and survival.

A Comparison between Chemical and Natural Dispersion of a North Sea Oil-spill

ABSTRACT The application of dispersants to an oil-slick is a key remediation tool and thus understanding its effectiveness is vital. Two in situ oil slicks were created in the North Sea (off the coast of The Netherlands), one left to natural processes whilst dispersant (Slickgone NS) was applied to the other. GC-MS analysis of seawater from the surface slick, and at 1.5 and 5 m below the slick, revealed only two samples with measurable hydrocarbons (221 ± 92 μg ml−1 seawater), from the surface of the “Slickgone Dispersed” oil-slick ~25.5 hours after oil-slick formation, which was likely due to environmental conditions hindering sampling. Additionally, 16S rRNA gene quantitative PCR and amplicon analysis revealed extremely limited growth of obligate hydrocarbonoclastic bacteria (OHCB), detected at a relative abundance of &amp;lt;1×10-6 %. Furthermore, the Ecological Index of Hydrocarbon Exposure (EIHE) score, which quantifies the proportion of the bacterial community with hydrocarbon-biodegradation potential, was extremely low at 0.012 (scale of 0 – 1). This very low abundance of hydrocarbon-degrading bacteria at the time of sampling, even in samples with measurable hydrocarbons, could potentially be attributed to nutrient limitation (~25.5 hours after oil-slick creation total inorganic nitrogen was 3.33 μM and phosphorus was undetectable). The results of this study highlight a limited capacity for the environment, during this relatively short period, to naturally attenuate oil.